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Abstract:

A fluorescence spectrophotometer having an excitation double
monochromator, a coaxial excitation/emission light transfer module, and
an emission double monochromator. Each monochromator includes a pair of
holographic concave gratings mounted to precisely select a desired band
of wavelengths from incoming broadband light without using other optical
elements, such as mirrors. Selected excitation light is directed into a
sample well by a light transfer module that includes a coaxial excitation
mirror positioned to direct excitation light directly to the bottom of a
well of a multi-well plate. Fluorescence emission light that exits the
well opening is collected by a relatively large coaxial emission mirror.
The collected emission light is wavelength selected by the emission
double monochromator. Selected emission light is detected by a
photodetector module.

Claims:

1. A reflection light transfer module including:(a) an input mirror,
positioned substantially coaxial with an area to be illuminated, for
directing incoming light to illuminate the area; and(b) an output mirror,
positioned substantially coaxial with the area to be illuminated and in
reflective alignment with the input mirror, for collecting and focusing
light emitted by the area upon illumination.

Description:

CROSS-REFERENCE TO RELATED APPLICATION

[0001]This application is a continuation application of allowed U.S.
application Ser. No. 10/658,363 filed Sep. 8, 2003, which claims benefit
of the priority of U.S. application Ser. No. 09/552,803 filed Apr. 20,
2000, now U.S. Pat. No. 6,654,119, which claims the benefit of priority
from U.S. Provisional Application Ser. No. 60/130,438, filed Apr. 21,
1999 the entire contents of all of which are incorporated herein by
reference.

TECHNICAL FIELD

[0002]This invention relates to wavelength scanning fluorescence
spectrophotometers using dual grating monochromators, but not optical
filters, to select excitation and emission wavelengths of light and to
detect and quantify simultaneous fluorescence emission from multiple
fluorophores in the same sample.

BACKGROUND

Definitions

[0003]1) Fluorescence: The result of a multi-stage process of energy
absorption and release by electrons of certain naturally occurring
minerals, polyaromatic hydrocarbons and other heterocycles.

[0004]2) Excitation: photons of energy, e=hvexc, are supplied by a
light source and absorbed by an outer electron of a fluorophore, which is
elevated from the ground state, S0, to an excited electronic singlet
state, S'1.

[0005]3) Excited State Lifetime: An excited electron remains in the
singlet state for a finite period, typically from 1 to 20 nanoseconds,
during which the fluorophore undergoes a variety of changes including
conformational changes and alterations in the interaction with solvent.
As a result of these changes, the energy of the S'1 singlet electron
partially dissipates to a relaxed singlet excited state, S'1, from
which fluorescence emission of energy occurs, returning the electron to
the ground state, S0.

[0006]4) Emission: photons of energy, e=hvem are released from an
excited state electron, which returns the fluorophore to the ground
state. Owing to energy loss during the excited state lifetime, the energy
of these photons is lower than that of the exciting photons, and the
emitted light is of longer wavelength. The difference in the energy (or
wavelengths) is called the Stoke's shift and is an important feature in
the selection of a dye for use as a label or in a probe. The greater the
Stoke's shift, the more readily low numbers of photons can be
distinguished from background excitation light.

[0007]5) Fluorophores: Fluorescent molecules are generally referred to as
fluorophores. When a fluorophore is utilized to add color to some other
molecule, the fluorophore is called a fluorescent dye and the combination
is referred to as a fluorescent probe. Fluorescent probes are designed
to: 1) localize and help visualize targets within a specific region of a
biological specimen, or, 2) respond to a specific stimulus.

[0008]6) Electromagnetic Spectrum: The entire spectrum, considered as a
continuum, of all kinds of electric and magnetic radiation, from gamma
rays, having a wavelength of 0.001 Angstroms to long waves having a
wavelength of more than 1,000,000 kilometers and including the
ultraviolet, visible and infrared spectra.

[0009]7) Fluorescence Spectrum: Unless a fluorophore is unstable
(photobleaches), excitation and emission is a repetitive process during
the time that the sample is illuminated. For polyatomic molecules in
solution, discrete electronic transitions are replaced by broad energy
bands called the fluorescence excitation and fluorescence emission
spectra, respectively.

[0010]8) Monochromator: A device which admits a wide spectral range of
wavelengths from the electromagnetic spectrum via an entrance aperture,
and, by dispersing wavelengths in space, makes available at an exit
aperture only a narrow spectral band of prescribed wavelength(s). Optical
filters differ from monochromators in that they provide wavelength
selection through transmittance of selected wavelengths rather than
through spatial dispersion. A second distinguishing feature of a
monochromator is that the output wavelength(s), and in many cases, the
output spectral bandwidth, may be continuously selectable. Typically, the
minimal optical components of a monochromator comprise: (a) an entrance
slit that provides a narrow optical image; (b) a collimator which ensures
that the rays admitted by the slit are parallel; (c) some component for
dispersing the admitted light into spatially separate wavelengths; (d) a
focusing element to re-establish an image of the slit from selected
wavelengths; and, (e) an exit slit to isolate the desired wavelengths of
light.

[0011]In a monochromator, wavelength selection is achieved through a drive
system that systematically pivots the dispersing element about an axis
through its center. Slits are narrow apertures in a monochromator which
may have adjustable dimensions. Slits effect selection of the desired
wavelength(s) and their dimensions may be adjustable.

[0012]9) Double Monochromator: Two monochromators coupled in series. The
second monochromator accepts wavelengths of light selected by the first
and further separates the prescribed wavelengths from undesired
wavelengths.

[0013]10) Wavelength Scanning: Continuous change of the prescribed output
wavelength(s) leaving the exit slit of a monochromator. In a
spectrophotometer, wavelengths of the electromagnetic spectrum are
scanned by the excitation monochromator to identify or prescribe the
wavelength(s) at which a fluorophore is excited; wavelength scanning by
the emission monochromator is used to identify and detect the
wavelength(s) at which a fluorophore emits fluorescent light. In
automated fluorescence spectrophotometers, wavelength scanning by the
excitation and emission monochromators may be performed either separately
or concurrently (synchronous scanning).

[0014]11) Area Scanning: Area scanning is distinct from wavelength
scanning and is the collective measurement of local fluorescence
intensities in a defined two dimensional space. The result is an image,
database or table of intensities that maps fluorescence intensities at
actual locations in a two dimensional sample. At its simplest, area
scanning may be a photograph made with a camera in which all data are
collected concurrently. Alternatively, the sample may be moved past a
detector which measures the fluorescence in defined sub-areas of a
sample. The collected information creates a matrix which relates
fluorescence intensity with position from which an image, table or
graphical representation of the fluorescence in the original sample can
be created.

[0015]12) Fluorescence Detectors:

[0016]Five elements of fluorescence detection have been established
through laboratory use of fluorophores during the last two decades: (a)
an excitation source, (b) a fluorophore, (c) some type of wavelength
discrimination to isolate emission photons from excitation photons, (d)
some type of photosensitive response element that converts emission
photons into a recordable form, typically an electronic signal or a
photographic image, and, (e) a light tight enclosure to restrict ambient
light.

[0017]Fluorescence detectors are primarily of four types, each providing
distinctly different information: (a) Cameras resolve fluorescence as
spatial coordinates in two dimensions by capturing an image: [a] as a
photographic image on highly sensitive film, or, [b] as a reconstructed
image captured on arrays of pixels in a charge coupled device (CCD). (b)
Fluorescence microscopes also resolve fluorescence as spatial coordinates
in two or three dimensions. Microscopes collect all of the information
for an image for a prescribed visual field at the same time without any
movement of either the sample or the viewing objective. A microscope may
introduce qualitative estimation of fluorophore concentration through use
of a camera to capture an image in which case the measure is a function
of exposure time. (c) Flow cytometers measure fluorescence per biological
cell in a flowing liquid, allowing subpopulations within a mixture of
cells to be identified, quantitated and in some cases separated. Flow
cytometers cannot be used to create an image of a defined area or perform
wavelength scanning. The excitation light source is invariably a laser
and wavelength discrimination is accomplished through some combination of
tunable dye lasers and filters. Although these instruments may employ
photomultiplier tubes to detect a measurable signal, there are no flow
cytometers that employ monochromators for wavelength scanning. (d)
Spectrofluorometers (spectrophotometer(s)) typically employ a PMT to
detect fluorescence but can measure either: [a] the average current
evoked by fluorescence over time (signal averaging), or, [b] the number
of photons per unit time emitted by a sample (photon counting).

[0018]Fluorescence spectrophotometers are analytical instruments in which
a fluorescent dye or probe can be excited by light at specific
wavelengths, and, concurrently, have its emitted light detected and
analyzed to identify, measure and quantitate the concentration of the
probe. For example, a piece of DNA may be chemically attached, or
labeled, with fluorescent dye molecules that, when exposed to light of
prescribed wavelengths, absorb energy through electron transitions from a
ground state to an excited state. As indicated above, the excited
molecules release excess energy via various pathways, including
fluorescence emission. The emitted light may be gathered and analyzed.
Alternatively, a molecule of interest may be conjugated to an enzyme
which can convert a specific substrate molecule from a non-fluorescent to
a fluorescent product following which the product can be excited and
detected as described above.

[0019]The ranges of excitation and emission wavelengths employed in a
fluorescence spectrophotometer typically are limited to the ultraviolet
and visible portions of the electromagnetic spectrum. For the purposes of
fluorescence detection, useful dyes are those which are excited by, and
emit fluorescence at, a few, narrow bands of wavelengths within the near
ultraviolet and visible portions of the electromagnetic spectrum. Desired
wavelengths for excitation of a specific fluorescent molecule may be
generated from:

[0020]1) a wide band light source by passing the light through a series of
bandpass filters (materials which transmit desired wavelengths of light
and are opaque to others), or cut-on filters (materials which transmit
all wavelengths longer or shorter than a prescribed value,

[0021]2) a narrow band light source such as a laser, or,

[0022]3) an appropriate monochromator.

[0023]For a wide band light source, the light to which a fluorescent dye
is exposed is typically isolated through bandpass filters to select a
desired wavelength from the ultraviolet or visible spectrum for use in
excitation. In monochromator-based instruments, the wavelength of choice
is obtained after light from the source has been dispersed into a
spectrum from which the desired wavelength is selected. Whatever the
light source, the fluorescence emission is typically isolated through
bandpass filters, cut-on filters, or emission monochromators to select a
desired wavelength for detection by removal of all light of any
wavelengths except the prescribed wavelengths. Most fluorescence
detection involves examination of specimens that are in a liquid phase.
The liquid can be contained in a glass, plastic or quartz container which
can take the form of, for example: an individual cuvette; a flow-through
cell or tube; a microscope slide; a cylindrical or rectangular well in a
multiwell plate; or silicon microarrays which may have many nucleic acids
or proteins attached to their surfaces. Alternatively, the liquid can be
trapped in a two-dimensional polyacrylamide or agarose gel. In each of
these cases, light which has already passed through the optical filters
to select the correct wavelengths for excitation illuminates the sample
in the container or gel; concurrently, emitted light is also collected,
passed through a second set of optical filters to isolate the wavelengths
of emission, and then detected using a camera, or photosensor.

[0024]The optical filters used in fluorescence detectors present
characteristics that limit the sensitivity, dynamic range and flexibility
of fluorescence detection, including: light absorption which causes a
loss of efficiency through the system; inherent auto-fluorescence, which
produces a high background signal; transmission of other wavelengths
outside the wavelengths of desired bandpass which, in turn limits both
sensitivity and dynamic range. Optical filters must be designed and
manufactured to select for discrete ranges of wavelengths ("center-width
bandpasses") which limits fluorescence detection to the use of compounds
which are excited and emit at wavelengths appropriate for those filters.
Development of a new fluorescent dye with unusual spectral properties may
necessitate design of a new excitation/emission filter pair.

[0025]To increase efficiency in fluorescence cuvette spectrophotometers as
well as to provide continuous selection of wavelengths, it has been known
to use grating or prism-based monochromators to disperse incoming light
from an excitation source, select a narrow band of excitation wavelengths
and, separately, to select an emission wavelength. Gratings come in many
forms but are etched with lines that disperse broadband light into its
many wavelengths. A monochromator typically includes a light-tight
housing with an entrance slit and an exit slit. Light from a source is
focused onto the entrance slit. A collimating mirror within the housing
directs the received beam onto a flat optical grating, which disperses
the wavelengths of the light onto a second collimating mirror which in
turn focuses the now linearly dispersed light onto the exit slit. Light
of the desired wavelength is selected by pivoting the grating to move the
linear array of wavelengths past the exit slit, allowing only a
relatively narrow band of wavelengths to emerge from the monochromator.
The actual range of wavelengths in the selected light is determined by
the dimensions of the slit. The process of continuous selection of a
narrow band of wavelengths from all wavelengths of a continuous spectrum
is referred to as wavelength scanning and the angle of rotation of the
dispersing optical grating with respect to the entrance and exit slits
correlates with the output wavelength of the monochromator. In order to
more precisely select the wavelengths of excitation and fluorescence
detection, it has been known to use two gratings in each monochromator to
enhance wavelength selection for both the excitation and emission light
in a fluorescence spectrophotometer. While the monochromators potentially
eliminate the need to use optical filters for wavelength selection and
free the scientist from the limitations of filters, their use imposes
other limitations on instrument sensitivity and design. For example,
monochromators having the configurations described above have the
disadvantage of requiring at least four mirrors and two dispersing
elements, along with associated light blocking entrance and exit slits.
Consequently, such devices have been relatively complex and comparatively
inefficient compared to filter based instruments.

[0026]Analysis of multiple samples in multi-well plates is a highly
specialized use of fluorescence spectrophotometers. Typically, the
excitation light is introduced into a well from a slight angle above the
well in order to allow the majority of the fluorescence emission light
from the sample within a well to be collected by a lens or mirror.
However, as the number of wells per plate is increased (e.g., from 96
wells per plate to in excess of 9600 per plate), this side illumination
configuration becomes disadvantageous, since most of the incoming
excitation light strikes the side of the well rather than the sample.
Since such wells typically have black side walls, much of the excitation
light is lost.

[0027]As mentioned above, one method employed to overcome the limitations
of side illumination configurations has been use of an optical fiber to
guide the excitation light to an illumination end of the fiber directly
positioned over a well. A second bundle of fibers is employed to collect
light from the well and transmit it to the PMT. In a variation of this
design, a bifurcated optical fiber positioned above a microwell has been
used to carry light both into and out of the well. However, optical
fibers typically introduce absorption losses and may also auto-fluoresce
at certain wavelengths. Accordingly, such a solution is not particularly
efficient.

[0028]Another approach has been to use multi-well plates with transparent
bottoms, and exposing a sample within a well to excitation light from the
bottom while collecting emission light from the open top. While this
approach has value in some circumstances, light is lost from absorption
as well as from light scattering by the plastic at the well bottom.

[0029]Additionally, the transparent plate material may itself
auto-fluoresce. In addition, well-to-well optical reproducibility of the
well bottom material has not been achieved, which has limited the ability
to correlate measurements on a well-to-well or plate-to-plate basis.
Accordingly, such a solution has proven to be less efficient than
illuminating and collecting light from the same side of a sample.

[0030]Examples of such prior art using fiber optic light paths include a
single unit fluorescence microtiter plate detector (the "Spectromax
GEMINI") introduced in 1998 by Molecular Devices, which employs a hybrid
combination of single grating monochromators, filters, mirrors and
optical fibers, and the "Fluorolog-3" a modular instrument and the
"Skin-Sensor", a unitized instrument, produced by Instruments SA, both of
which employ bifurcated fiber optic bundles to conduct light from an
excitation monochromator and to collect light from a sample after which
it is transmitted to the excitation monochromator.

[0031]It should be noted that microtiter plate applications of
fluorescence monochromators are also limited to microwell plates with
384, 96 or fewer wells; that is, 1536-well microplates as well as
"nanoplates" containing 2500 wells, 3500 wells and even 9600 wells cannot
be used with the current fiber-optic/monochromator based instruments.
Detectors for such plates typically use lasers and filters combined with
confocal microscopy.

[0032]For the large number of applications involving glass microscope
slides, polyacrylamide gels or standard 96-well, 384-well and 1536-well
microwell plates, it would be desirable to have a fluorescence
spectrophotometer that provides high efficiency, enables high precision
continuous excitation and emission wavelength selection, provides
significantly greater dynamic range, eliminates the use of optical
filters and optical fibers (i.e., light paths do not pass through any
optical materials other than air), and has a highly efficient structure
for both guiding the excitation light onto a sample and collecting the
emission light from the sample in a microtiter well or on a two
dimensional surface such as a glass microscope slide, polyacrylamide gel,
silicon microarray, or other solid surfaces.

[0033]In general, the measurement of fluorescent light intensity, the
luminescence, is defined as the number of photons emitted per unit time.
Fluorescence emission from atoms or molecules can be used to quantitate
the amount of an emitting substance in a sample. The relationship between
fluorescence intensity and analyte concentration is:

F=kQeP0(1-10.sup.[Ebc])

where F is the measured fluorescence intensity, k is a geometric
instrumental factor, Qe is the quantum efficiency (photons
emitted/photons absorbed), Po is the probability of excitation which
is a function of the radiant power of the excitation source, ε is
the wavelength dependent molar absorptivity coefficient, b is the path
length and c is the analyte concentration. In previous applications, the
above equation was simplified by expanding the equation in a series and
dropping the higher terms to give:

F=kQeP0(2.303*ε*b*c)

[0034]In the past, this relationship was acceptable because fluorescence
intensity appeared to be linearly proportional to analyte concentration.
The equation fails, however, to provide for true comparison of the
fluorescence intensities of different fluorophores because measurement of
fluorescence intensity is highly dependent upon k, the geometric
instrumental factor.

[0035]Different types of detectors vary in both the time period during
which a measurement is made and the speed at which each can discriminate
between photons, characteristics which can be of critical importance when
comparing the luminosity of two fluorophores. Consider two fluorescent
dyes that differ only in that the excited state lifetime of one is
tenfold longer than that of the excited state lifetime of the second
fluorophore (e.g., 1 nanosecond and 10 nanoseconds, respectively). When
detected using photographic film exposed for a defined exposure time, the
dye with the shorter lifetime would clearly appear brighter on the
exposed film. However, if a detector employing continuous excitation were
used which could not discriminate between photons at a high enough
frequency, the fluorophore with the shorter excited state lifetime could
actually be emitting far more photons but the detector could erroneously
indicate that the fluorescence intensity of the two dyes was the same.

[0036]Fluorescence is detected in spectrophotometers through generation of
photocurrent in an appropriate photosensitive device such as a
photomultiplier tube or other photosensing device, both of which are
characterized by low levels of background or random electronic noise. For
this reason, fluorescent emission processes are best characterized by
Poisson statistics and fluorescence can be measured through either photon
counting or signal averaging:

[0037]Photon counting is a highly sensitive technique for measurement of
low levels of electromagnetic radiation. In photon counting detection,
current produced by a photon hitting the anode of a photomultiplier tube
with sufficient energy to begin an avalanche of electrons is tested by a
discriminator circuit to distinguish between random electronic noise and
true signal. At such light levels, the discreteness of photons dominates
measurement and requires technologies that enable distinguishing
electrical pulses that are photon-induced from dark-current impulses that
originate in the detector (e.g., a photomultiplier tube) from other
causes.

[0038]In previous applications of photon counting, the dynamic range of
detection was restricted by the ability of the detector to discriminate
between photons closely spaced in time. Additionally, the signal to noise
ratio in photon counting is also a function of the light intensity.
Assume a steady light flux incident on a photocathode producing m
photoelectrons per second. During any one second, the light incident on
the photocathode is, on average, m photoelectrons with a standard
deviation of m1/2. The signal to noise ratio in such measurements
is:

S/N=m/m1/2=m1/2 (1)

[0039]Depending upon their frequency and energy, individual photoelectrons
can be counted with a detector of sufficient gain but the precision of
any measurement can never be better than the limit imposed by equation
(1). In its simplest form, a practical photon counting instrument
consists of a fast amplifier and a discriminator set to a low threshold
relative to the input, typically -2 mV, which has been found empirically
to correspond to the optimum compromise between susceptibility to
electrical pickup and operating the photomultiplier at excessive gain.

[0040]Theoretically less sensitive than photon counting and with greater
sensitivity to electronic drift, signal averaging uses photocurrent as a
direct measure of the incident light signal. The noise associated with
the photocurrent Ik, taking the system bandwidth (frequency of
response), B, into account, is given by the shot noise formula:

S/N=(Ik/2eIkB)1/2 (2)

where e is the electronic charge. The forms of equations 1 and 2 are
similar since they refer to the same phenomenon and predict essentially
the same result. In contrast to photon counting, in which the signal is
inherently digitized and its dynamic range limited by the speed of the
timer counter, in equation 2 the signal is taken as a continuous variable
of the photocurrent and it is possible to obtain a much larger dynamic
range. In practice, however, the analog-to-digital conversion process
severely limits the dynamic range owing to the slow response times
associated with A/D converters having more than 16-bit resolution.

[0041]In general purpose fluorescence detection instruments, the light
source can be a quartz halogen lamp, a xenon lamp or similar gas
discharge lamp, a photodiode or one of many types of lasers. Typically
the sample is exposed to continuous illumination which maintains a
relatively stable percentage of the total number of fluorophores in an
excited state. In these instruments, the cross-sectional dimension of the
sample which is illuminated is principally determined by slits. In more
complex instruments, including any using imaged light, confocal optics or
point source illumination, the exciting light beam is shaped and focused
by lenses and mirrors onto a single point and a single focal plane in the
sample.

SUMMARY

[0042]According to various embodiments of the present invention, a
wavelength and area scanning fluorescence spectrophotometer is provided
that includes an excitation double monochromator, a coaxial
excitation/emission light transfer module, an emission double
monochromator a high speed timer-counter circuit board and a precision
x-y-z mounting table for use in positioning a sample relative to the
focal plane of the exciting light. Operations of each are directed and
coordinated through a timer-counter board.

[0043]Each monochromator includes a pair of holographic concave gratings
mounted to precisely select a desired band of wavelengths from incoming
broadband light. Selected excitation light is directed into a sample well
or onto a two dimensional surface such as a polyacrylamide gel or
microscope slide by a light transfer module that includes a coaxial
excitation mirror positioned to direct excitation light directly into a
well of a multi-well plate or onto a particular area of a gel, microscope
slide or microarray. Emitted light that exits the sample is collected by
a relatively large front-surfaced mirror. The collected emission light is
wavelength selected by the emission double monochromator. Both
monochromators contain three precision matched slits that are positioned
to restrict unwanted wavelengths while simultaneously creating a "near
point" source of the desired wavelengths for the succeeding stage of the
optical path. Emission light that has been isolated in this way is
projected onto the photodetector module which converts the received
energy into a digital representation of the fluorescence intensity of the
sample.

[0044]One embodiment includes a fluorescence spectrophotometer system
having a light source; a first double monochromator operating to separate
and output selected wavelengths of light from the light source as
excitation light; a light transfer module for directing substantially all
of the excitation light directly onto a sample, and for collecting,
focusing, and directing fluorescence from the sample as emission light; a
second double monochromator operating to separate and output selected
wavelengths of the emission light; and a photodetector and analyzer for
detecting the selected wavelengths of emission light and outputting an
indication of such detection.

[0045]Another embodiment includes a double monochromator having an
entrance slit for accepting light; a first optical grating positioned to
intercept and disperse the accepted light from the entrance slit; a first
selection slit positioned to intercept at least part of the dispersed
light from the first optical grating and select and pass a narrowed range
of wavelengths from such dispersed light; a second optical grating
positioned to intercept and disperse the passed light from the first
selection slit; and a second selection slit positioned to intercept at
least part of the dispersed light from the second optical grating and
select and pass a narrowed range of wavelengths from such dispersed
light.

[0046]Yet another embodiment includes a light transfer module having an
input mirror, positioned coaxially with an area to be illuminated, for
directing incoming light to illuminate the area; and an output mirror,
positioned coaxially with the area to be illuminated and in reflective
alignment with the input mirror, for collecting, focusing, and directing
light emitted by the area upon illumination.

[0048]The details of one or more embodiments of the invention are set
forth in the accompanying drawings and the description below. Other
features, objects, and advantages of the invention will be apparent from
the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

[0049]FIG. 1 is a schematic diagram of an embodiment of a fluorescence
spectrophotometer system.

[0050]FIG. 2A is a schematic diagram of another embodiment of a double
monochromator.

[0051]FIG. 2B is a schematic diagram showing a tension band actuator
mechanism for pivoting the gratings of the double monochromator shown in
FIG. 2A.

[0052]FIG. 3 is a schematic diagram of one embodiment of the light
transfer module shown in FIG. 1.

[0053]FIG. 4 is a schematic diagram of a simplified version of a
fluorescence spectrophotometer in accordance with the present invention.

[0054]FIG. 5A shows a first alternative dual path embodiment of the
invention.

[0055]FIG. 5B shows a second alternative dual path embodiment of the
invention.

[0056]Like reference numbers and designations in the various drawings
indicate like elements.

DETAILED DESCRIPTION

[0057]FIG. 1 is a schematic diagram of an embodiment of a fluorescence
spectrophotometer system in accordance with the present invention. A
broadband light source 100 illuminates a mirror 102, which is suitably
curved to focus the light onto a double monochromator 104. In alternative
configurations, a narrow band light source such as a photodiode or a
laser can be substituted for the broadband light source but used with the
same monochromator configuration.

[0058]Light of the desired wavelength is passed by the double
monochromator 104 to a light transfer module (LTM) 106. The LTM 106
directs the excitation light from monochromator 104 onto sample 108,
which can be, for example, one well of a microwell plate or one lane of a
1-D polyacrylamide gel. Any resulting fluorescence emitted by the sample
is collected by LTM 106 which directs the light to the entrance slit of
emission double monochromator 110. The emission monochromator is adjusted
to pass wavelengths from the emission spectrum of the fluorophore(s) in
the sample and directs those wavelengths to photodetector 112, which
measures the energy of the emitted light.

[0059]Photodetector 112 may be any suitable photosensitive device,
including but not limited to a photomultiplier tube, a phototransistor,
or a photodiode. The electronic output of photodetector 112 is applied to
an automatic processing unit 114, which generates a signal indicating
detection of the selected emission which is stored in a numerical form
suitable for further analysis. The automatic processing unit 114 may be,
for example, a personal computer having a data collection interface to
the spectrophotometer system. In general, all of the elements of the
optical pathways in this instrument, including double monochromators 104
and 110 and the LTM 106, should be isolated in light-tight boxes coated
internally with non-fluorescent absorptive material to minimize
reflectances and light from other sources such a room light.

[0060]FIG. 2A is a schematic diagram of an embodiment of a double
monochromator in accordance with the present invention. The illustrated
configuration may be used for either the excitation double monochromator
104 or the emission double monochromator 110.

[0061]Broadband light is introduced through a slit 200 in the double
monochromator and reflects off the front surface of a first holographic
concave grating 202 that is pivotable around an axis 203. Use of front
surface reflection enhances the efficiency of the double monochromator by
avoiding absorption of the light within an optical support structure,
such as in a rear-surface reflection glass mirror. Use of a concave
grating allows the light to be dispersed into selectable wavelengths
without the use of supplemental collimating mirrors. This design makes it
possible to eliminate two collimating mirrors per grating used in
conventional dual monochromator instruments. Use of a holographic grating
also reduces astigmatic aberrations and thus decreases the amount of
light of unwanted wavelengths ("stray light").

[0062]Each double monochromator has three slits: an entrance slit, through
which light first enters the monochromator; an internal selection slit;
and, an exit selection slit, through which light leaves the
monochromator. The first concave grating 202 reflects the wavelengths of
the incoming light as a first spatially dispersed beam 204. Each
wavelength of this first spatially dispersed beam 204 is reflected at a
unique angle relative to other wavelengths. By pivoting the first concave
grating 202 around its axis 203, a desired band of wavelengths can be
directed through the internal selection slit 206 within the monochromator
housing.

[0063]The selected range of wavelengths 208 is then reflected off a second
holographic concave grating 210 that is pivotable about an axis 211. The
second concave grating 210 reflects the wavelengths of the first
spatially dispersed beam 204 as a second spatially dispersed beam 212. By
pivoting the second concave grating 210 around its axis 211, a desired
narrow band of wavelengths 214 can be directed through an exit selection
slit 216.

[0064]The widths of selection slits 206 and 216 determine the selected
wavelengths of light leaving the monochromator. Wider slits allow more
light energy to pass through a monochromator, but the light includes a
broad range of wavelengths; narrower slits reduce the amount of light
passing through a monochromator but narrow the selected range of
wavelengths. The use of wider slits increases the sensitivity of
detection, which is beneficial in measurements of total fluorescence in
an area or volume as would be the case in measurements made in
microwells. By contrast, the use of narrow slits increases the spatial
resolution which is beneficial in discriminating between different
fluorophores at particular locations as is the case with bands separated
in a polyacrylamide gel. That is, the smaller the slit dimensions, the
smaller the area of detection at the sample. In the case of laser light
sources and pinhole slits, the area of excitation at any given moment is
a point that corresponds to a particular data pair representing
fluorescent intensity and position.

[0065]It is a general rule in optical systems that each optical component
reduces the efficiency of light throughput. Advantages of the
configuration shown in FIG. 2A include, but are not limited to,
elimination of collimating mirrors for redirecting light within the
double monochromator, such as is the case with traditional
monochromators. In the present embodiment, only two light directing
elements (the first and second holographic concave gratings 202, 210) are
required, thus improving overall light efficiency.

[0066]In the configuration illustrated in FIG. 2A, the first concave
grating 202 and the second concave grating 210 are pivoted oppositely and
in tandem by a suitable mechanism. A mechanism according to one
embodiment uses a tension band actuator mechanism for pivoting the
gratings 202, 210, as shown in FIG. 2B. The gratings 202, 210 are mounted
coaxially with pivot wheels 250, 252, respectively. A lever arm 254 is
connected to pivot wheel 250 at one end and a screw drive mechanism 256
at the other end. Lever arm 254 is moved along threaded rod 258 as the
rod is rotated by motor 260.

[0067]Lever arm 254 rotates pivot wheel 250 as it travels along rod 258.
Pivot wheel 250 is connected to pivot wheel 252 by a tension band 262.
According to the present embodiment, tension band 262 is a 0.003 inch
stainless steel band. Tension band 262 causes the pivot wheel 252 and
grating 210 to counter-rotate in tandem with pivot wheel 250 and grating
202. A spring 264 connected between a housing and pivot wheel 252
maintains tension in tension band 262. The position of the grating can be
determined and controlled by a microcontroller connected to pivot wheel
sensor 266 and screw drive sensor 268.

[0068]FIG. 3 is a schematic diagram of one embodiment of the LTM 106 shown
in FIG. 1. Excitation light enters the LTM 106 through an entrance
aperture whereupon it is directed, either with or without one or more
mirrors 300, to a coaxial excitation mirror 302. The coaxial excitation
mirror 302 may be flat, or have some curvature to focus or disperse light
as desired. The coaxial excitation mirror 302 is positioned to direct the
excitation light onto the sample 108. More particularly, the coaxial
excitation mirror 302 may be positioned somewhat off-axis with respect to
sample 108, but should be positioned so that substantially all of the
excitation light strikes the sample to achieve maximum illumination.

[0069]In one application, each well of a multi-well plate can be
positioned beneath the coaxial excitation mirror 302 by X-Y translation
of either the LTM 106 or of the multi-well plate. In another application
involving monochromators equipped with optional microscope optics,
different regions of intact biological cells that have been mounted on
glass slides or culture plates can be separately imaged by using optical
elements in the light path and by positioning the sample beneath the
coaxial excitation mirror 302 by X-Y-Z translation of either the LTM 106
or of the glass slides or culture plates. Any fluorescence emission from
one or more fluorophores in a sample is collected by a coaxial emission
mirror 304. The coaxial emission mirror 304 must be concave so as to
focus and direct the emission light, either directly or by one or more
light directing mirrors 306, out of an exit port of the LTM 106. In the
embodiment shown in FIG. 3, the emission light exits the LTM 106 from the
same side that the excitation light enters the LTM 106. However,
different placements of the entrance and exit ports can be used by
suitable placement of light directing 300, 306.

[0070]The coaxial placement of the excitation mirror 302 and the emission
mirror 304 ensures that a high percentage of the excitation light is
directed onto the sample within a well 108, and that a high percentage of
the fluorescence light emitted from the well opening is collected for
analysis.

[0071]In the preferred embodiment, all of the mirrors within the LTM 106
comprise front, or "first" surface mirrors. Such mirrors have a
reflective material, such as aluminum, coated onto the surface of a
substrate, such as glass or ceramic onto which light is directed. The
coating serves as the reflective surface, so that light does not
penetrate the substrate, as in an ordinary second surface mirror. Such
first surface mirrors are substantially more efficient.

[0072]It will be understood by one of ordinary skill in the art that a
number of different reflecting mirrors may be used to direct the light
within the LTM 106, as needed. However, it is desirable that the number
of such reflecting surfaces be minimized in order to improve efficiency
of the LTM 106. In the preferred embodiment, the coaxial excitation
mirror 302 is an elliptical mirror approximately 6×9 millimeters in
dimension, while the coaxial emission mirror 304 is approximately 75
millimeters in diameter. Other dimensions can be used and generally will
vary with the dimensions of the overall instrument.

[0073]FIG. 4 is a schematic diagram of a simplified version of a
fluorescence spectrophotometer in accordance with an embodiment of the
present invention. In this embodiment, excitation wavelengths are
selected from a broadband light source 100 by means of an excitation
double monochromator 104. The excitation light is directed by a coaxial
excitation mirror 302 to a sample within a well 108. Fluorescence
emissions are collected and focused by a coaxial emissions mirror 304
that is in reflective alignment with the coaxial excitation mirror 302.
The coaxial emissions mirror 304 directs the collected and focused light
into an emission double monochromator 110. The emission double
monochromator 110 selects a desired emission wavelength and directs that
wavelength to a photodetector 112 for counting and analysis. This
embodiment minimizes the number of reflective surfaces within the LTM
106.

[0074]One aspect of the embodiment shown in FIG. 4 is that its compact
configuration allows for use of the instrument as a dual path
spectrophotometer. That is, a sample can be excited from both the open
side of a well 108 or from the bottom side of the well.

[0075]FIG. 5A shows a first alternative dual path embodiment of the
invention. One or both of the light source 100 and the excitation double
monochromator 104 optionally can be translated from their normal position
to a position below a multi-well plate such that excitation light
impinges upon a bottom-illumination coaxial excitation mirror 302', which
directs excitation light through the transparent bottom substrate of a
well 108. (If the light source is not translated, light directing mirrors
may be interposed to direct light through the excitation double
monochromator 104). Fluorescence emissions emanating from the opening of
the well 108 are collected by a coaxial emission mirror 304. When
configured for bottom illumination, the top illumination coaxial
excitation mirror 302 can be removed from the light path in order to
maximize the amount of fluorescent light collected by the emission mirror
304. Alternatively, the top-illumination excitation mirror 302 (not shown
in FIG. 5A) can be left in place, as shown in FIG. 4. In either case, the
bottom-illumination excitation mirror 302' is in direct alignment (as
opposed to reflective alignment) with the emissions mirror 304.

[0076]FIG. 5B shows a second alternative dual path embodiment of the
invention. In this configuration, for bottom illumination, a set of one
or more redirection mirrors 303A, 303B, are interposed into the normal
light path from the excitation double monochromator 104 in order to
intercept the excitation light. The intercepted excitation light is
redirected to impinge upon a bottom-illumination coaxial excitation
mirror 302', which directs the excitation light through the transparent
bottom substrate of a well 108. The top illumination coaxial excitation
mirror 302 may be left in place, as shown, or moved out of position when
the redirection mirrors 303A, 303B, are moved into position. Such
movement may be accomplished, for example, by translational movement of a
carriage on which all four mirrors 302, 303A, 303B, 302', are mounted,
into or out of the page. Again, the bottom-illumination excitation mirror
302' is in direct alignment (as opposed to reflective alignment) with the
emissions mirror 304.

[0077]In all of the configurations shown in FIGS. 4, 5A, and 5B,
additional redirection mirrors may be used as desired to suitably guide
light to desired locations within the instrument.

[0078]According to an embodiment, a timer-counter board that operates at a
frequency in excess of 100 MHz is used. The discriminator module can
respond to photons at frequencies as high as 30 MHz. The dynamic range of
this system ranges from 0 to more than 30×106 photons, thereby
eliminating the dynamic range limitations which previously restricted the
use of photon counting in fluorescence detection. If the observed current
from the PMT is greater than a prescribed threshold established at the
discriminator, a 5 volt electrical pulse having a duration of
approximately 2 nanoseconds is produced; the timer counter circuit
enumerates these pulses as a function of time, that is, establishes a
direct quantitation for photons per unit time.

[0079]In a spectrophotometer according to one embodiment, a pre-cast
polyacrylamide gel containing fluorescently labeled nucleic acids or
proteins was placed on a flat plate in the positioning mechanism of the
present invention directly under the LTM. With the excitation and
emission monochromators set at wavelengths suitable for the fluorescent
labels, the gel was moved back and forth under the LTM until all of the
gels area had been traversed. At each point of this travel, a fluorescent
reading was made and stored as a two dimensional array representing the
fluorescent emission at each point of the gel. The distance between the
points was adjusted to yield the best response for a given data
acquisition time. In the actual experiments, the gel was also scanned as
"lanes" representing the path of electrophoresis from the sample well at
the top of the gel to the base of the gel because the fluorophores in an
electrophoresis gel are arrayed in a line rather than as a point. Each
such lane was scanned from the sample well to the bottom of the gel to
achieve a substantial reduction in overall data collection time. As a
reference for background, a blank lane was scanned and the data
subtracted on a point-by-point basis from the corresponding data for
lanes containing fluorophores. The corrected data were then analyzed in
two ways. In one analysis, an image was constructed of the original gel
which was compared to standard laboratory photographs of the same gel for
evaluation of standard gel parameters such as migration distance,
separation of molecules and concentration of molecular species as
determined separately by the digital image and the film. In the other
analysis, a "densitometry" plot equivalent to those made for gel lanes
from autoradiography films using flat bed scanning detectors was created.
From the database relating fluorescence intensity of a lane, the center
of each fluorescent band was identified and a cross sectional graph of
fluorescent intensities as a function of migration distance was prepared.
From the use of gels prepared with different but known amounts of the
same fluorescent labeled nucleic acids, a standard curve establishing
lower and upper limits of sensitivity, resolution, and overall dynamic
range for gel detection were determined.

[0080]The "square intensity point spread function" and the "long
penetration depth" properties of one and two photon absorption processes
have been recognized as important features in future developments in
fluorescence detection. Both are accomplished by focusing a femtosecond
short pulse laser onto a focal plane in a sample to be studied for
fluorescence. In a spectrophotometer according to another embodiment, a
laser beam from an appropriate laser was substituted for the quartz
halogen or xenon light source used for excitation. The laser beam was
used to illuminate the entrance slit of the excitation monochromator.
Additional modifications where needed in some cases included, for
example, the use of one or two pinhole slits rather than the standard
rectangular slits, and the insertion of an objective lens to focus the
light after it had passed through the monochromator. The fluorescence
emission was collected through the light transfer module as previously
and the fluorescence measured as a function of time, or in the cases of
image formation and area scanning, as a function of time and position of
the light transfer module over the sample as described in gel analysis,
above. In this configuration, a pinhole exit slit on the excitation
monochromator was used to image light onto a specimen and each point of
the image used to excite fluorescence. Moving the sample position in an
x-y-z fashion, enabled scanning of areas of the sample to create an image
or database. For confocal microscopy, two pinhole slits were required as
described below. For multi-photon applications, only a single pinhole
slit was used as the exit slit of the excitation monochromator. For
two-photon excitation, a microlens array could be used if needed to focus
the beam for high transmittance. In general, the configuration was
epi-fluorescent although in certain polarization applications, excitation
was from the bottom and collection of light from the top.

[0081]In yet another embodiment, the present invention was applied in the
creation of a scanning fluorescence polarization detector. Fluorescent
molecules in solution, when excited with plane-polarized light, will emit
light back into a fixed plane (i.e., the light remains polarized) if the
molecules remain stationary during the fluorophore's period of excitation
(excited state lifetime). Molecules in solution, however, tumble and
rotate randomly, and if the rotation occurs during the excited state
lifetime and before emission occurs, the planes into which light is
emitted can be very different from the plane of the light used for the
original excitation.

[0082]The polarization value of a molecule is proportional to its
rotational relaxation time, which by convention is defined as the time
required for a molecule to rotate through an angle of 68.5°.
Rotational relaxation time is related to the solution viscosity (η),
absolute temperature (T), molecular volume (V), and the gas constant (R):

Polarization value ∝ Rotational Relaxation
Time = 3 η V RT

[0083]If viscosity and temperature are constant, the polarization is
directly related to molecular volume (molecular size), which, in general,
correlates well with molecular weight. Changes in molecular volume result
from several causes, including degradation, denaturation, conformational
changes, or the binding or dissociation of two molecules. Any of these
changes can be detected as a function of changes in the polarization
value of a solution. Specifically, a small fluorescent molecule which
rotates freely in solution during its excited state lifetime can emit
light in very different planes from that of the incident light. If that
same small fluorophore binds to a larger molecule, the rotational
velocity of the small molecule decreases and the effect is detected as a
decrease in the polarization value. Measurement of the effect requires
excitation by polarized light which can be obtained using a laser or
through light selection using a polarizing filter which only transmits
light traveling in a single plane. In one experiment, the light of a
defined wavelength obtained from the excitation monochromator of the
present invention was further refined by passing the light through a
polarizing filter designated the "polarizer", to obtain monochromatic,
plane-polarized light for excitation (for the present purposes designated
"vertically polarized light"). Concurrently, the light path for
collecting the emitted light was similarly modified by introduction of a
second polarizing filter, designated the "analyzer", which could be
rotated to positions either vertical or horizontal to the plane of the
exciting light. When a fluorescent sample is solution was introduced into
the light path between the "polarizer" and the "analyzer", only those
molecules which were oriented properly to the vertically polarized plane
absorbed light, became excited, and subsequently emitted light. By
rotating the analyzing filter, the amount of emitted light in the
vertical and horizontal planes could be measured and used to assess the
extent of rotation of the small fluorescent molecule in the solution
before and after binding to a larger molecule.

[0084]In yet another application, the invention was utilized in confocal
microscopy, a method for eliminating one of the fundamental difficulties
of fluorescence microscopy, namely the reduction in spatial resolution at
the focal plane of the microscope owing to out-of-focus light. A
spectrophotometer according to another embodiment was used to create a
novel confocal microscope from the embodiment essentially as described
under laser excitation above to focus a light image on whole cell mounts
and achieve both multiple and single photon excitation of the fluorescent
labels in a sample. Fluorophores in planes out of the focus were not
illuminated and did not fluoresce.

[0085]In confocal imaging, apertures were used in both the excitation and
emission light paths in order to focus a cone of light through the
specimen and in the emission light path in order to eliminate scattered
and out-of-plane fluorescence. The development of mode locked dye lasers
has made simultaneous multiphoton excitation practical because such
lasers are capable of delivering the available excitation energy to a
focal spot in very brief pulses and with sufficient energy to achieve two
photon excitation. In multiphoton imaging, the focal spot provided by the
laser excites a sufficiently small volume that, when used in conjunction
with the light transmission module of this invention, makes it possible
to collect all emission light without a second pinhole slit on the
emission side. No emission aperture changes were necessary.

[0086]A number of embodiments of the present invention have been
described. Nevertheless, it will be understood that various modifications
may be made without departing from the spirit and scope of the invention.
For example, the double monochromators of the invention can be used in
other types of instruments, and the LTM may be used in a filter-based
spectrophotometer or other optical instruments. As a further example, the
LTM may be used to direct input light to an area to be illuminated and
efficiently collect, focus, and direct light emitted (e.g., either by
reflection or by fluorescence) from the illuminated area. Accordingly,
other embodiments are within the scope of the following claims.